Development of a HPLC method for the determination of cyclosporin-A in rat blood and plasma using naproxen as an internal standard

An isocratic reversed phase high-performance liquid chromatographic (HPLC) method with ultraviolet detection at 205 nm has been developed for the determination of cyclosporin-A (CyA) in rat blood and plasma. Naproxen was successfully used as an internal standard. Blood or plasma samples were pretreated by liquid–liquid extraction with diethyl ether. The ether extract was evaporated and the residue was reconstituted in acetonitrile–0.04 M monobasic potassium phosphate buffer (pH 2.5) solvent mixture. After washing with n-hexane, 30 μl of the reconstituted solution was injected into HPLC system. Good chromatographic separation between CyA and internal standard peaks was achieved by using a stainless steel analytical column packed with 4 μm Nova-Pak Phenyl material. The system was operated at 75 °C using a mobile phase consisting of acetonitrile–0.04 M monobasic potassium phosphate (pH 2.5) (65:35 v/v) at a flow rate of 1 ml/min. The calibration curve for CyA in rat blood was linear over the tested concentration range of 0.0033–0.0166 M with a correlation coefficient of 0.989. For rat plasma, the range of the concentrations tested were between 0.002 and 0.0166 M and showed linearity with a correlation coefficient of 0.953. The intra- and inter-run precision and accuracy results were 1.24–21.87 and 3.1–12.23%, respectively. The low volume of blood or plasma needed (200 μl), simplicity of the extraction process, short run time (5 min) and low injection volume (30 μl) make this method suitable for quick and routine analysis.     

LC determination of dinitrosopiperazine in simulated gastric juice

A simple and specific reversed phase HPLC method for the determination of dinitrosopiperazine in simulated gastric juice using UV detection was reported. The chromatographic resolution of the analyte and the internal standard isosorbide dinitrate was performed without extraction from the gastric juice on a reversed phase ODS column. Isocratic elution was carried out with methanol–0.02 M sodium dihydrogen phosphate (60:40 v/v, pH 3.0) at a flow rate of 1.0 ml min⁻¹ with UV detection at 238 nm. The calibration graph was linear over the concentration range 0.072–2.88 μg ml⁻¹ of dinitrosopiperazine with minimum detectability (S/N=2) of 0.01 μg ml⁻¹ (8×10⁻⁸ M). Inter-day and intra-day precisions calculated as% RSD were in the range 0.32–0.38% and 0.19–0.25% respectively. Inter-day and intra-day accuracies calculated as% error were in the range 0.18–0.21 and 0.08–0.11% respectively. The proposed method was successfully applied to the study of the possible in–vivo production of DNPZ under the standard nitrosation conditions recommended by WHO.     

The estimation of paracetamol and its major metabolites in both plasma and urine by a single high-performance liquid chromatography assay

Many analytical methods exist for the assay of paracetamol in biological fluids, including colorimetry with chemical derivatization, direct spectrophotometry, chromatographic methods and immunoassays. Their development has been largely driven by the needs of clinical toxicology requiring the rapid, reliable and highly specific estimation of paracetamol in plasma samples to determine the need for antidote therapy. However, for in vivo metabolism studies, a specific assay method which can provide measurements of paracetamol and its metabolites in both plasma and urine is desired. A reversed-phase HPLC method with UV detection at 254 nm was developed to fulfil these requirements. The assay involves minimum sample preparation with a relatively short run time. The solvent system involves a simple isocratic elution with a composition of 0.1 M potassium dihydrogen orthophosphate—acetic acid—propan-2-ol, (100:0.1:0.75, v/v/v). The reproducibility of the assay was high with an inter-assay RSD of 0.2–1.7% for urinary paracetamol concentrations of 5–500 μg ml⁻¹ and 0.1–3.3% for plasma concentrations between 5 and 25 μg ml⁻¹. A similarly high degree of precision was found for the glucuronide, sulphate, cysteine and mercapturate metabolites of paracetamol. The same assay can be used to analyse both plasma and urine samples and thus was employed for studies on the metabolism of paracetamol in healthy subjects and in patients with various diseases.     

A pharmacological examination of venom from the Papuan taipan (Oxyuranus scutellatus canni).

The Papuan taipan (Oxyuranus scutellatus canni) is the third most venomous terrestrial snake in the world, however, little is know about the pharmacology of the venom. In the chick biventer cervicis muscle, venom (10 μg/ml) abolished nerve-mediated twitches (time to 90% inhibition (t₉₀) 44±5 min, n=9). This inhibition was unaffected by prior incubation of the venom with the phospholipase A inhibitor 4-bromophenacyl bromide (4-BPB; 0.72 mM) (t₉₀ 48±7 min, n=8). The mouse phrenic nerve diaphragm preparation displayed greater sensitivity to venom (10 μg/ml) (t₉₀ 25±1 min, n=6). In the chick biventer muscle, venom (10 μg/ml) significantly inhibited responses to acetylcholine (1 mM) and carbachol (20 μM), but not KCl (40 mM), indicating activity at post-synaptic nicotinic receptors. Venom (10 μg/ml) did not affect direct muscle stimulation. Venom (3–30 μg/ml) produced dose-dependant contractions of the guinea-pig ileum. Contractile responses were significantly inhibited by indomethacin (1 μM) or prior incubation of the venom with 4-BPB (0.72 mM) indicating involvement of a PLA component. In rat phenylephrine (0.3 μM) precontracted aortae, venom (3–100 μg/ml) produced endothelium-independent relaxation which was unaffected by prior incubation of venom (30 μg/ml) with 4-BPB (0.72 mM). In anaesthetised rats, 10 μg/kg (i.v.) venom produced rapid respiratory and cardiovascular collapse while 5 μg/kg (i.v.) venom produced only a small transient decrease in mean arterial blood pressure. Prior administration of 5 μg/kg (i.v.) venom enabled subsequent administration of 10 and 100 μg/kg (i.v.) venom without respiratory or cardiovascular collapse. Further work is required to identify specific toxins with the above pharmacological activity.     

Determination of clotrimazole in mice plasma by capillary electrophoresis

In addition to its antifungal activity, clotrimazole attracts interest as an anti-inflammatory drug. In order to correlate this effect with plasma concentrations in mice, a capillary electrophoretic method was developed. Sample preparation was carried out by protein precipitation using methanol. Quantification of clotrimazole was achieved by means of capillary electrophoresis using ketoconazole as an internal standard (IS). The background electrolyte (BGE) composed of a Tris buffer solution (100 mM, pH 3.0, adjusted with acetic acid) and methanol (8:2, v/v). Injection was carried out electrokinetically with 10 kV over a time period of 20 s. A special rinsing procedure utilizing a sequence of a SDS/methanol solution, a sodium hydroxide solution, water and BGE, was applied to enhance the reproducibility. With this procedure, an intermediate precision (day-to-day precision) of the area ratios of clotrimazole and IS of 5.0% for 0.5 μg ml⁻¹ and 2.6% for 10 μg ml⁻¹ was obtained. In summary, with the described capillary zone electrophoresis (CZE) method it is possible to handle small sample volumes of 60 μl, to detect clotrimazole concentrations of 0.3 μg ml⁻¹ (limit of detection), and to quantify clotrimazole down to concentrations of 0.5 μg ml⁻¹ (limit of quantification).     

Analysis of brefeldin A and the prodrug breflate in plasma by gas chromatography with mass selective detection

Breflate is a water soluble prodrug developed to facilitate parenteral administration of the investigational antineoplastic agent brefeldin A (BFA). Previously, using analytical methods based upon reversed-phase high performance liquid chromatography (HPLC) with low wavelength UV detection or gas chromatography (GC) with electron capture detection following derivatization with heptafluorobutyrylimidazole, it was demonstrated that breflate undergoes rapid and efficient conversion to BFA following bolus i.v. injection in mice and dogs. However, plasma concentrations of the drug and prodrug achieved during the administration of nontoxic doses of breflate to beagle dogs as a 72 h continuous i.v. infusion were undetectable (<0.1 μg ml⁻¹) by these methods. The sensitivity and specificity required for therapeutic drug level monitoring were achieved by GC with selected-ion mass spectrometry (MS) detection. Breflate, BFA and 1-eicosanol, the latter added to the sample as an internal standard (IS), were extracted from plasma into tert-butyl methyl ether (TBME) and esterified with trifluoroacetic anhydride. Methanol was added to the reaction mixture to effect the convenient removal of excess reagent as the volatile methyl ester during evaporation of the solvent. The residual material was analyzed directly upon reconstitution by capillary GC with automated splitless injection. Electron-ionization (70 eV) MS detection was performed by sequentially scanning ions at m/z 58, 202 and 325. The lowest concentration of either analyte quantified with acceptable reproducibility, as defined by an inter-day R.S.D. of about 20%, was near 10 ng ml⁻¹ in plasma using a sample volume of 100 μl. The assay has proven to be sufficiently sensitive, specific and reproducible for the routine analysis of pharmacokinetic specimens acquired during IND (investigational new drug)-directed toxicology studies in dogs.     

Simultaneous determination of nikethamide and lidocaine in human blood and cerebrospinal fluid by high performance liquid chromatography

Nikethamide and lidocaine are often requested to be quantified simultaneously in forensic toxicological analysis. A simple reversed-phase high performance liquid chromatography (RP-HPLC) method has been developed for their simultaneous determination in human blood and cerebrospinal fluid. The method involves simple protein precipitation sample treatment followed by quantification of analytes using HPLC at 263 nm. Analytes were separated on a 5 μm Zorbax Dikema C₁₈ column (150 mm × 4.60 mm, i.d.) with a mobile phase of 22:78 (v/v) mixture of methanol and a diethylamine–acetic acid buffer, pH 4.0. The mean recoveries were between 69.8 and 94.4% for nikethamide and between 78.9 and 97.2% for lidocaine. Limits of detection (LODs) for nikethamide and lidocaine were 0.008 and 0.16 μg/ml in plasma and 0.007 and 0.14 μg/ml in cerebrospinal fluid, respectively. The mean intra-assay and inter-assay coefficients of variation (CVs) for both analytes were less than 9.2 and 10.8%, respectively. The developed method was applied to blood sample analyses in eight forensic cases, where blood concentrations of lidocaine ranged from 0.68 to 34.4 μg/ml and nikethamide ranged from 1.25 to 106.8 μg/ml. In six cases cerebrospinal fluid analysis was requested. The values ranged from 20.3 to 185.6 μg/ml of lidocaine and 8.0 to 72.4 μg/ml of nikethamide. The method is simple and sensitive enough to be used in toxicological analysis for simultaneous determination of nikethamide and lidocaine in blood and cerebrospinal fluid.     

High-throughput multi-analyte screening for renal disease using capillary electrophoresis

End-state renal disease (ESRD) affects 300 000 people in the United States each year. A large percentage of these individuals (20%) die within the first year after diagnosis. Current methods of determining renal function rely on the measurement of a single marker using slow and frequently non-specific colorimetric methods. In this report, capillary zone electrophoresis was used to perform a multi-analyte assay for markers of renal function in urine. This method tested for creatinine (Cr), creatine (Cn), uric acid (UA), and p-aminohippuric acid (PAH) levels. The limits of detection (S/N=3) were found to be 5 μM for Cr, 0.75 μM for Cn, and 1.5 μM for UA and PAH. Linear ranges were determined to be 5–500 μM for Cr, 0.75–500 μM for Cn, and 1.5–250 μM for UA and PAH. These ranges included the expected concentrations of the markers in human urine after 50-fold dilution. This screening method proved to be a simple and fast way to perform a high throughput analysis for multiple renal function indicators.     

Residue determination of two co-administered antibacterial agents — cephalexin and colistin — in calf tissues using high-performance liquid chromatography and microbiological methods

Residues of two antibacterial agents, cephalexin and colistin, co-administered by intramuscular injection to calves, were quantified in four different tissues (muscle, fat, liver and kidney) by column switching HPLC and by a microbiological method. For cephalexin assay, tissue samples with cephradin as internal standard were homogenized in a 5% trichloroacetic acid solution and filtrates were injected onto a concentration pre-column filled with LiChroprep RP-18 (25–40 μm). A clean-up step was incorporated by flowing a mobile phase (methanol—0.01 M phosphate buffer (pH 3.0); 15:85, v/v) through the enrichment column before elution on a LiChrospher RP-18e (5 μm) column with a methanol—phosphate buffer (30:70, v/v) at a flow rate of 1 ml min⁻¹. Spectrometric detection was at 260 nm. An additional “off-line” washing step of extracts with methylene chloride was operated to achieve higher selectivity in the case of liver and kidney samples. The limit for quantitative assay was 0.045 μg g⁻¹ with relative standard deviations in the range 5–8% and recoveries within 70%.

For microbiological assay of colistin, samples were homogenized in 0.1 M hydrochloric acid–acetonitrile mixtures (3:1, v/v, for kidney and liver; 3:2, v/v, for fat and muscle). The supernatants were assayed by the cylinder plate method after evaporation to dryness under vacuum. Bordetella bronchiseptica ATCC 4617 was chosen as test organism. After a 3-h diffusion step at room temperature, the medium was incubated at 37°C for 18 h and then the diameter of the growth inhibition zones was measured. Sensitivity reached 0.10–0.15 μg g⁻¹. Results from the analysed samples over a 7–28 day period after drug administration show that no cephalexin was found at concentrations higher than the quantitation limit in the four test tissues and that colistin was found in muscle (injection site only) for 15 days and in kidney for 21 days.     

Synthesis, organotropism and hepatocellular uptake of two tritium-labeled epimers of dihydromicrocystin-LR, a cyanobacterial peptide toxin analog

J. A. O. , S. E. , A. M. and J. E. . Synthesis, organotropism and hepatocellular uptake of two tritium-labeled epimers of dihydromicrocystin-LR, a cyanobacterial peptide toxin analog. Toxicon, 28, 1439–1446, 1990.—Two tritium-labeled epimers of dihydromicrocystin-LR, a derivative of the cyanobacterial peptide hepatotoxin microcystin-LR, were synthesized by reduction with sodium boro[³H]hydride and purified with reversed-phase liquid chromatography. The epimers were hepatotoxic in mice; the i.p. ₅₀ was 120–135 μg/kg. They were concentrated in the liver and to some extent in the intestine and the kidney after an i.v. injection. Freshly isolated rat hepatocytes showed a rapid uptake of both epimers. The cellular uptake of the epimers was almost complete within 5 min at concentrations 1 μM (0.5 μM dihydromicrocystin-LR + 0.5 μM microcystin-LR) and 4 μM (0.5 μM + 3.5 μM). The uptake of the earlier eluting epimer was about three times higher than that of the later eluting epimer.     

Determination of gabapentin in human plasma and urine by high-performance liquid chromatography with UV-vis detection

A simple and reliable high-performance liquid chromatographic (HPLC) method with UV–vis detection has been developed and validated for the determination of gabapentin (GBP) in human plasma and urine. The clean up of the sample was carried out by solid-phase extraction with C18-cartridge. After the clean up procedure, the samples were pre-column derivatizated with 1,2-naphthoquinone-4-sulphonic acid sodium salt (NQS). A chromatographic separation was achieved on a C18 column with a mobile phase consisting of acetonitrile and 10 mM orthophosphoric acid (pH 2.5) with isocratic elution (35:65). Baclofen was used as an internal standard (I.S.). The method developed for GBP was linear over the concentration range of 0.05–5.0 μg/ml and 0.1–10.0 μg/ml for plasma and urine, respectively. The method is precise (relative standard deviation, R.S.D. <4.05%) and accurate (relative mean error, RME <0.15%); mean absolute recoveries were 72.21% for plasma and 72.73% for urine.     

Involvement of 5-hydroxytryptamine in the blood pressure changes evoked by electrical stimulation of the median raphe nucleus

Electrical stimulation of the median raphe nucleus (MRN) in urethane-anaesthetised rats decreased systemic blood pressure at low intensities of stimulation (5–25 μA) and increased it with higher intensities (20–150 μA). Prazosin (0-5-5.0 μg/Kg i.v.) dose-dependently attenuated pressor responses concurrently with a reduction in the responsiveness of peripheral alpha-adrenoceptors to phenylephrine (500ng i.V.). Methlothepin (5–10 μg/Kg i.v.) abolished depressor responses and reduced the pressor effects without altering the response to phenylephrine. Ketanserin (5–10 μg/Kg i.v.) abolished depressor changes and potentiated pressor responses. High doses (20–200 μg/Kg) produced a decrease in pressor responses but correspondingly lowered BP and reduced the response to phenylephrlne.

The results suggest the presence of 5-HT-containlng links between the MRN and the peripheral cardiovascular effector systems.     

Nitrotyrosine attenuates the hemodynamic effects of adrenoceptor agonists in vivo: relevance to the pathophysiology of peroxynitrite

Peroxynitrite, which attenuates catecholamine-mediated hemodynamic responses in vivo, nitrates free tyrosine residues to form the specific product, 3-nitro- -tyrosine. The chemical structure of 3-nitro- -tyrosine is similar to that of the endogenous catecholamines. Therefore, 3-nitro- -tyrosine may interfere with catecholamine hemodynamic function in vivo. The hemodynamic responses produced by norepinephrine (1-4 μg/kg, i.v., n = 6), epinephrine (0.5-4 μg/kg, i.v., n = 7), phenylephrine (1-8 μg/kg, i.v., n = 5), and isoproterenol (100-400 ng/kg, i.v., n = 5) were attenuated, while the hemodynamic responses produced by arginine vasopressin (50-250 ng/kg; i.v., n = 5) were unaffected following the administration of 3-nitro- -tyrosine (2.5 μmol/kg, i.v.) in pentobarbital-anesthetized rats. These results demonstrate substantial and selective attenuation of the hemodynamic effects produced by - and β-adrenoceptor agonists, raising the possibility that 3-nitro- -tyrosine may play a role in the hemodynamic dysfunction associated with inflammatory conditions in which the formation of peroxynitrite is favored.     

Quantitation of vancomycin and its crystalline degradation product (CDP-1) in human serum by high performance liquid chromatography

The delayed clearance of vancomycin results in accumulation of vancomycin crystalline degradation product, CDP-1, in the bodies of renally impaired patients. The 2 isomers, CDP-1-M (major) and CDP-1-m (minor), of CDP-1 are antibiotically inactive but cross-react with some immunoassays that use polyclonal antibodies resulting in falsely elevated results. A high performance liquid chromatographic (HPLC) method was developed to quantitate vancomycin and CDP-1 in the serum of renal patients. After solid phase extraction of 200 μl serum, the separation of vancomycin, the 2 isomers of CDP-1 and the internal standard (cefazolin) was accomplished by gradient HPLC on a reversed phase C18 column with detection at 210 nm. Linearity was established from 1 to 25 and 25 to 100 μg ml⁻¹ vancomycin and 1 to 25 μg ml⁻¹ CDP-1. Coefficients of variation for vancomycin and CDP-1 were 3.3–8.6% (n=10) and 2.8–5.2% (n=8).     

Spectrophotometric determination of oxytetracycline in pharmaceutical preparations using sodium molybdate as analytical reagent

A spectrophotometric method is proposed for the determination of oxytetracycline in pharmaceutical preparations. The method is based on the measurement of the absorbance of the molybdate—oxytetracycline complex at 404 nm (pH 5.50; μ = 0.1 M; 20°C). The composition of the complex (1:1) was determined by the application of the spectrophotometric methods of Job and Bent—French (pH 5.50; λ = 390 nm; μ = 0.1 M). The relative stability constant (K′ = 10^4.6) of the complex was obtained by the methods of Sommer and Nash (pH 5.50; λ = 390 nm; μ = 0.1 M; 20°C). The molar absorptivity of the complex was 9.5 × 10³ l mol⁻¹ cm⁻¹. Beer's law was obeyed over the concentration range 2.48–34.78 μg ml⁻¹. The relative standard deviation RSD (n = 10) was 0.27–0.39%. The method proposed can be applied to the assay of oxytetracycline in capsules. The detection limit of oxytetracycline is 2.5 μg ml⁻¹.     

Tolerance to the vascular effect of a novel nitric oxide-donating vasodilator, FK409

We investigated whether tolerance develops to the vasorelaxant effects of a new vasodilator, (±)-(E)-4-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenamide (FK409), in isolated canine coronary artery strips and to its hypotensive effect in rats, and whether FK409 activates soluble guanylate cyclase isolated from vascular tissues in the absence of -cysteine. No tolerance to FK409 (0.46 nM to 0.46 μM or 1–1000 μg/kg, i.v.) or cross-tolerance between FK409 and glyceryl trinitrate was demonstrated in vitro and in vivo experiments, whereas the tolerance to glyceryl trinitrate (0.44 nM to 4.4 μM or 1–1000 μg/kg, i.v.) was marked in both conditions. In addition, FK409 (0.1–10 μM) activated soluble guanylate cyclase without -cysteine, but glyceryl trinitrate (1–100 μM) required the addition of -cysteine (5 mM) for the activation of the enzyme. The results suggest that FK409 may be advantageous compared to tolerance-producing nitrates currently in clinical use, and that this property of FK409 is probably due to its independence of a sulfhyhydryl donor.     

Determination of imperatorin in rat plasma by reversed-phase high-performance liquid chromatography after oral administration of Radix Angelicae dahuricae extract

A simple high-performance liquid chromatographic (HPLC) method has been developed for the determination of imperatorin in rat plasma and applied to a pharmacokinetic study in rats after administration of Radix Angelicae dahuricae extract. After addition of fluocinonide as an internal standard (IS), plasma samples were extracted with diethyl ether. HPLC analysis of the extracts was performed on a Diamonsil C18 analytical column using methanol–water (70:30, v/v) as the mobile phase. The UV detector was set at 254 nm. The standard curve was linear over the range 0.04–4.0 μg/mL. The lower limit of quantification was 0.04 μg/mL. The HPLC method developed could be easily applied to the determination and pharmacokinetic study of imperatorin in rat plasma after giving the animals Radix Angelicae dahuricae extract.     

Alternative high-performance liquid chromatographic assay for p-aminohippuric acid (PAH): effect of aging on PAH excretion in the isolated perfused rat kidney

Para-aminohippuric acid (PAH), an indicator of renal plasma flow, is a commonly used marker of organic anion transport by the kidney. An analytical method for PAH using HPLC was developed. The method is simple, fast and requires a minimum amount of organic solvent. Sample preparation involved protein precipitation with zinc sulfate. Para-amino benzoic acid was utilized as an internal standard (IS). Chromatography was performed using a reversed-phase phenyl column with UV detection at a wavelength of 254 nm. Mobile phase consisted of 0.1 M acetic acid and acetonitrile (99:1) at a flow rate of 1 ml/min. The assay was validated over a standard concentration range from 1 to 25 μg/ml. Accuracy, precision, reproducibility and specificity of the method was established with coefficients of variation <10%. The method was sensitive and showed linear response in peak height ratio (analyte:IS) over the concentration range studied (r²>0.99). The assay was used to study the effect of aging on PAH excretion in the isolated perfused rat kidney model. Experiments were conducted in kidneys from young (2–3 months, n=6), adult (6–9 months, n=5) and aged (12–16 months, n=3) male Sprague–Dawley rats at an initial drug concentration of 20 μg/ml. Significant differences in kidney function (e.g. glomerular filtration rate and glucose reabsorption) were observed in aged kidneys. Despite a 5-fold reduction in glomerular filtration rate, PAH renal clearance (kidney weight-corrected) decreased by only 2-fold in aged (2.2±0.42 ml/min per gram) compared to young (4.6±0.70 ml/min per gram, P<0.05) rats. Furthermore, renal excretion ratio was significantly higher in aged rats (27±8.0 vs. 15±5.0, P<0.05). These preliminary findings challenge the ‘Whole Nephron Hypothesis’ that assumes parallel reductions in renal filtration and secretory capacity secondary to disease or aging.     

Comparative evaluation between capillary electrophoresis and high-performance liquid chromatography for the analysis of florfenicol in plasma

A capillary electrophoresis (CE) and a reversed phase high-performance liquid chromatography (RP-HPLC) method with UV detection have been developed for florfenicol analysis in plasma samples. The suitabilities of both methods for quantitative determination of florfenicol were approved through validation specification, such as linearity, precision, selectivity, accuracy, limit of detection and quantification. The capillary electrophoresis (CE) and high-performance liquid chromatography (HPLC) assay were compared by analyzing a series of plasma samples containing florfenicol in different concentrations using the two methods. The extraction procedure is simple and no gradient elution or derivatization is required. Furthermore, the analysis time of the CE method is two times shorter than the respective parameter in HPLC and solvent consumptions is considerably lower. The calibration curve were linear to at least 0.05–10 μg/ml (r = 0.9998) and 0.1–10 μg/ml (r = 0.9998) for CE and HPLC, respectively. The separation efficiency are good for both methods. The detection limits for florfenicol were 0.015 μg/ml with CE and 0.03 μg/ml with HPLC and CE method gave lower value, even though UV detector was applied in the both cases. The both methods were selective, robust and reliable quantification of florfenicol and can be useful for clinical and biomedical investigations.     

Simultaneous separation and determination of active components in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS

A simple and rapid isocratic LC/MS coupled with electrospray ionization (ESI) method for simultaneous separation and determination of adenine, hypoxanthine, adenosine and cordycepin in Cordyceps sinensis (Cs) and its substitutes was developed. 2-Chloroadenosine was used as internal standard for this assay. The optimum separation for these analytes was achieved using the mixture of water, methanol and formic acid (85:14:1, v/v/v) as a mobile phase and a 2.0×150 mm Shimadzu VP-ODS column. Selective ion monitoring (SIM) mode ([M+H]⁺ at m/z 136, 137, 268, 252 and 302) was used for quantitative analysis of above four active components. The regression equations were liner in the range of 1.4–140.0 μg ml⁻¹ for adenine, 0.6–117.5 μg ml⁻¹ for hypoxanthine, 0.5–128.5 μg ml⁻¹ for adenosine and 0.5–131.5 μg ml⁻¹ for cordycepin. The limits of quantitation (LOQ) and detection (LOD) were, respectively 1.4 and 0.5 μg ml⁻¹ for adenine, 0.6 and 0.2 μg ml⁻¹ for hypoxanthine, 0.5 and 0.1 μg ml⁻¹ for adenosine and cordycepin. The recoveries of four constituents were from 93.5 to 107.0%. The nucleoside contents of various types of natural Cs and its substitutes were determined and compared with this developed method.